Clostridium difficile is a spore-forming, gram-positive bacillus that produces exotoxins that are pathogenic to humans. C. difficile-associated disease (CDAD) ranges in severity from mild diarrhea to fulminant colitis and death. C. difficile typically has affected older or severely ill patients who are hospital inpatients or residents of long-term-care facilities. C. difficile is the major cause of pseudomembranous colitis and antibiotic associated diarrhea. C. difficile-associated disease occurs when the normal intestinal flora is altered, allowing C. difficile to flourish in the intestinal tract and produce a toxin that causes a watery diarrhea. One major cause for alteration of intestinal flora is the overuse of antibiotics. Repeated enemas, prolonged nasogastric tube insertion and gastrointestinal tract surgery also increase a person's risk of developing the disease. The overuse of antibiotics, especially penicillin (ampicillin), clindamycin and cephalosporins may also alter the normal intestinal flora and increase the risk of developing C. difficile diarrhea.
Toxigenic strains of C. difficile commonly produce two large toxins, an enterotoxin; toxin A (TcdA) and a cytotoxin; toxin B (TcdB), to which disease symptoms are attributed. They are expressed efficiently during growth of C. difficile in response to an environmental stimulus. Their activities modulate numerous physiological events in the cell and contribute directly to disease. In humans the two toxins cause diseases called pseudomembranous colitis and antibiotic associated diarrhea. Transmission occurs primarily in health care facilities, where exposure to antimicrobial drugs and environmental contamination by C. difficile spores are common (2, 3 and 4).
Toxin A and toxin B are encoded by genes tcdA and tcdB. Both have been sequenced and are found in single open reading frames. Together with three additional genes (tcdC, tcdD, tcdE), they form a 19.6 kb chromosomal pathogenicity locus (Paloc) (8). Both open reading frames are large, with tcdA spanning 8,133 nucleotides and tcdB being 7,098 nucleotides in length. FIG. 1 shows the genetic arrangement of the C. difficile Paloc. tcdD, renamed tcdR (Rupnik, M. et al., J. Med. Microbiol, 2005, 54: 113-117) is a proposed positive regulator, tcdE is a putative holin protein, and tcdC is a proposed negative regulator of toxin gene expression (Voth, D. E. et al., Clinical Microbiol. Reviews, 2005, 18: 247-263).
TcdA and TcdB are among the largest bacterial toxins reported, comparable in size to lethal toxin (TcsL) and hemorrhagic toxin (TcsH) of C. sordellii as well as alpha toxin (Tcns) of C. novyi (Voth, supra.). TcdA (308 kDa) and TcdB (270 kDa) are glucosyltransferases which inactivate small GTPases such as Rho, Rac and Cdc-42 within target cells (Voth, supra.). This inactivation causes disagreggation of the cellular cytoskeleton and alterations of other cellular processes which eventually lead to cell death (Voth, supra.). Both toxins use a highly conserved N-terminal domain (74% homology between TcdA and TcdB) to modify identical substrates. The proximal locations of tcdA and tcdB genes and the high sequence and functional homology between the two proteins inspired Von Eichel-Streiber to propose that the two genes may have arisen as the result of gene duplication (Knoop F. C. et al, Clin. Micro reviews, July 1993, 251-265).
TcdB also exhibits homology (85% homology and 74% identity) with lethal toxin (TscL) of C. sordellii, which glycosylates Ras, Rac, Rap and Ral. The major differences are found in the N terminus. These explain the differences in substrate specificity. TcdA is thought to be more similar in function to the hemorrhagic toxin (TcsH) of C. sordellii (Voth, supra.).
In early studies, it had been generally accepted that C. difficile toxigenic strains produced both toxin A and toxin B whereas nontoxigenic strains lacked both toxins (Rupnik et al. supra.; Lyerly et al., Clin. Micro. Rev., 1998, Jan., 1-18). Toxin variant strains were then discovered which failed to produce detectable toxin A, and yet produced toxin B (TcdA−/TcdB+). A third toxin (binary toxin CDT) has also been found in some C. difficile strains. Although the majority of binary toxin positive strains produce TcdA and TcdB (TcdA+TcdB+CDT+) some produce neither TcdA nor TcdB (TcdA−TcdB−CDT+). In the light of available data, C. difficile strains into toxigenic strains were classified as toxigenic if they produced at least one of the three known toxins, and nontoxigenic strains if they did not produce any of these three toxins (Rupnik et al., supra.).
While the primary work on TcdA and TcdB was carried out on toxins from the toxigenic reference strain VP1 10463, several genetic variants of these toxins now exist in clinical isolates (Voth et al., supra.). Two well-characterized strains which do not express toxin A (TcdA−TcdB+), 1470 and 8864, produce modified toxin B compared to VP1 10463. Strain 1470 produces a hybrid of toxins TcdB and TcsL. The strain produces TcdB-like cell contact and a TcsL-like enzymatic domain (morphological change and cell death like TcsL) (Voth, supra.; Chaves-Olarte E. et al, The Journal of biological chemistry, 1999, 274, no16, 11046-11052). As mentioned above, toxin B from reference strain 10463 inactivated small GTPases as Rho, Rac and Cdc-42. The impact is visible on electron microscopy with a modification of cellular aspect. Two types of cytopathic effects are described. The D-type is characterized by an arborized appearance of the cells whereas a spindle-like appearance is typical of the second type of cytopathic effect, the S-type (Mehlig, et al., FEMS Microbiol. Lett., 2001, 198:171-176). Toxin B of reference strain show D-type cytopathic effect as well as toxin A. Strains with lack of toxin A production, such as strain 1470 and strain 8864, produce toxin B with S-type cytopathic effect. Substrates for these toxins B are small GTPases Ras, Rac, Rap, Ral and Cdc-42. Both strains show variations in their toxin B gene (tcdB) compared to VP1 14063 tcdB gene. These variations explain the differences in substrate specificity. A difference in the N-terminal region of the tcdB of 1470 strain and VP1 10463 has been well documented (Von Eichel-Streiber et al, Mol Microbiol, 1995, 17: 313-321).
Another toxin B variant strain was discovered that produces functional toxin A. Thus, strain C34 is the first C. difficile strain that expresses a variant toxin B as 1470 and 8864, and a functional toxin A as reference type strain 14063 (Mehlig et al., supra.). This strain produces a toxin B with S-type cytopathic effect such as strain 1470 and 8864. C34 is the first C. difficile isolate coexpressing a D-type-inducing TcdA with an S-type-inducing TcdB molecule. The substrates of TcdA-C34 and the reference strain TcdA-10463 are identical (Rho, Rae and Cdc-42), and the substrates of TcdB-C34 and TcdA-1470 or 8864 are identical (Ras, Rac, Rap, Ral and Cdc-42). The tcdB sequence from C34 differs only in nucleotides from tcdB-1470 or 8864. Instead of having a deletion in tcdA that prevents toxin A production as strains 1470 and 8864, there is an inserted sequence in tcdA-C34. This small insertion does not have a negative effect on toxin A production. Nevertheless, in this strain, the S-type cytopathic effect on cells dominates over the D-type cytopathic effect (Mehlig et al., supra.).
To date, one variant strain has been described that produces a generally intact tcdB but a non-functional toxin B lacking a cytotoxic effect, and a functional toxin A having a cytotoxic effect. Toxinotyping data of this variant showed limited mutation in the Paloc and classified this strain in toxinotype IX (TcdA+/TcdB+/CDT+) (abstract, Maccannell et al, 2006). Recently, outbreaks of hypertoxigenic C. difficile strains have been reported in Canada and the United States. These isolates were positive for CDT binary toxin, had a deletion in the tcdC gene and produced greater amounts of toxins A and B (McDonald et al, New Engl. J. Med., December 2005, 353, no 23). The emergence of similar C. difficile isolates in the UK, Belgium and the Netherlands has also been described. The epidemic strain isolated in those countries was characterized as toxinotype III, North American PGEF 1 (NAP1), restriction endonuclease analysis group type B1 and PCR ribotype 027 (Kuijper E et al, document for European Centre for Disease prevention and Control, Emergence of Clostridium difficile-associated disease in Canada, the United State of America and Europe).
For C. difficile toxigenic strains, nucleotide sequence variations, deletions and duplications in the Paloc (tcdB and tcdA region) account for various types. A typing system has been developed which distinguishes the various types and classifies them as toxinotypes (1, 8, 9, 10, 11, 12, 13, 19). Toxinotyping involves detection of polymorphisms in the pathogenicity locus (Paloc) precisely in the tcdA and tcdB genes. There are now at least 24 toxinotypes (See Table 1). Strains in which the Paloc is identical to the reference strain VP1 10463 are referred as toxinotype 0. Not all variations of toxin genes affect toxin production. Strains of toxinotypes I-VII, TX, XII-XV and XVIII-XXIV produce both toxins A and B despite variations in their toxin genes (8, 11, 13, 19). Strains of toxinotype XI do not produce toxin A or B (13) whereas strains of toxinotypes VIII, X, XVI and XVII produce a functional toxin B but no toxin A (13). FIG. 2 describes well the relation between toxinotype and toxin expression. Strain 1470 belongs to toxinotype VIII and strain 8864 to toxinotype X. Most of the TcdA−/TcdB+ strains are known to belong to toxinotype VIII and produce a variant toxin B like strain 1470 while toxinotype X contains only strain 8864 (11).
TABLE 1Clostridium difficile toxinotypesToxinToxinotypeStrainStrain originproduction(1)0VP1 10463USAA+B+ CDT−IEX623BelgiumA+B+ CDT−IIAC008FranceA+B+ CDT−IIIaSE884Not availableA+B+ CDT+IIIbR10278Not availableA+B+ CDT+IIIcCH6230Not availableA+B+ CDT+IV55767BelgiumA+B+ CDT+VSE881FranceA+B+ CDT+VI51377BelgiumA+B+ CDT+VII57267BelgiumA+B+ CDT+VIII1470BelgiumA−B+ CDT−IX51680BelgiumA+B+ CDT+X8864EnglandA−B+ CDT+XI aIS58Not availableA−B− CDT+XI bR11402Not availableA−B− CDT+XIIIS25Not availableA+B+ CDT−XIIIR9367Not availableA+B+ CDT−XIVR10870EnglandA+B+ CDT+XVR9385Not availableA+B+ CDT+XVISUC36IndonesiaA−B+ CDT+XVIIJ9965JapanA−B+ CDT+XVIIIGAI00166KoreanA+B+ CDT−XIXTR13JapanA+B+ CDT−XXTR14JapanA+B+ CDT−XXICH6223USAA+B+ CDT−XXIICH6143USAA+B+ CDT−XXIII8785BelgiumA+B+ CDT+XXIV597BKuwaitA+B+ CDT+(1)A+ and B+ refers to production of toxin TcdA and TcdB; CDT+ refers to the presence of complete CDT locus.
The consensus sequence for the tcdB gene was determined using 6 available sequences in GenBank (See Appendix I). The first and seventh sequences in the tcdB alignment (SEQ ID NO: 1) (SEQ ID NO: 1) is are the reference strain VP1 14063 TcdA+/TcdB+. The second and third sequences in Appendix I (SEQ ID NOS 2 and 3, respectively) are two well-characterized TcdA−/TcdB+ strains (1470, second line and strain 8864, third line). The fourth line is another TcdA−/TcdB+ strain (5340) (SEQ ID NO: 4). The variant toxB and functional toxA strain C34 cluster 1-2 sequence (SEQ ID NO: 5) is shown in the fifth line, and the C. sordellii lethal toxin (TcsL) sequence (SEQ ID NO: 6) is shown in the sixth line as a specificity control. SEQ ID NO: 5 ends at nucleotide 1695. Thus, the six sequences in the alignment from nucleotides 1696 to 7095 (top to bottom) are SEQ ID NOS: 1, 2, 3, 4, 6, and 1, respectively. From nucleotides 7096 until the end of the sequence, SEQ ID NO: 1 is only shown once (top sequence), with the remaining sequences (top to bottom) being SEQ ID NOS: 1, 2, 3, 4, and 6, respectively. Certain regions of the tcdB gene are conserved among these different strains.
A positive culture for C. difficile without a toxin assay is not sufficient to make the diagnosis of C. difficile-associated disease. Thus, toxigenic C. difficile detection by a tissue culture cytotoxin assay is often considered the “gold standard.” However, this assay is time consuming, as it implies an incubation period of at least 24 h. The present invention provides a real-time PCR assay targeting the C. difficile toxin gene tcdB that is rapid, sensitive, and specific, and allows detection of C. difficile directly from clinical samples, such stool samples.